Combination therapies for inhibition of ttk protein kinase

ABSTRACT

Provided herein are methods of treating cancer using an effective amount of a compound represented by the formula: (I) or a pharmaceutically acceptable salt thereof and an effective amount of an immune checkpoint inhibitor. Also provided are compositions comprising the same compound represented by the formula shown above or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.

CROSS REFERENCE TO A RELATED APPLICATION

This reference claims priority to International Patent Application No.PCT/CA2017/050920, filed Aug. 1, 2017, which is incorporated herein byreference in its entirety.

BACKGROUND

CFI-402257 is a compound represented by the formula:

that inhibits TTK protein kinase (also known as Monopolar spindle 1 orMps1) activity. TTK is a core protein of the spindle assembly checkpoint(SAC), which is a signaling cascade that prevents chromosomemissegregation by arresting the cell cycle in mitosis until allchromosomes are properly attached to the mitotic spindle. Thus, the SACensures healthy proliferation and precise division in cells. Many cancercells have a weakened SAC response that may contribute to malignancy.However, more severe disabling of the SAC generates a level ofchromosome instability that exceeds the adaption capacity of cancercells, and is a potential anticancer strategy. Many tumors are shown toover-express TTK, which correlates with worse prognosis. Inhibition ofTTK activity in cancer cells causes severe chromosome missegregation,cell death and inhibited tumor growth.

CFI-402257 is presently undergoing a phase I clinical trial(ClinicalTrials.gov ID NCT02792465) with patients having advanced solidcancers. Given the potency and selectivity of CFI-402257 in inhibiting acritical mitotic checkpoint protein, it would be advantageous to furtherenhance the efficacy of this drug candidate in cancer treatment.

SUMMARY

It has now been found by the inventors of the present application thatthe administration of the compound CFI-402257 or a pharmaceuticallyacceptable salt thereof and an immune checkpoint inhibitorsynergistically treats cancer. See e.g., FIGS. 1D and 2D, which bothillustrate complete tumor regression in the syngeneic CT26 mouse coloncarcinoma model upon administration of a combination of the compoundCFI-402257 or a pharmaceutically acceptable salt thereof and the ratIgG2a anti-PD-1antibody. Importantly, when the animals in which completeregression have occurred are re-challenged by inoculation with the samecancer cells, tumors did not grow in any of these animals, therebyindicating that immunity to the cancer cells has been generated by theCFI-402257-anti-PD-1-antibody combination therapy. Furthermore, animalsthat are subjected to this combination therapy do not suffer from anysignificant body weight loss, thereby indicating that both agents atleast the administered dosages are well-tolerated.

Based on these results, provided herein are methods of treating cancerin a subject, by administering to the subject an effective amount of thecompound CFI-402257 or a pharmaceutically acceptable salt thereof and aneffective amount of an immune checkpoint inhibitor as described herein.

Also provided herein are pharmaceutical compositions comprising thecompound CFI-402257 or a pharmaceutically acceptable thereof and animmune checkpoint inhibitor as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive rat IgG2a PD-1 antibody (control).

FIG. 1B illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive 150 μg of rat IgG2a anti-PD-1 antibody on Days 0, 3, 6 and 10.

FIG. 1C illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive 6 mg/kg of CFI-402257 daily for 21 days.

FIG. 1D illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive the combination of 150 μg of rat IgG2a anti-PD-1 antibody onDays 0, 3, 6 and 10 and 6 mg/kg of CFI-402257 daily for 21 days.

FIG. 2A illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive rat IgG2a PD-1 antibody (control) in a duplicate experiment.

FIG. 2B illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive 150 μg of rat IgG2a anti-PD-1 antibody on Days 0, 3, 6 and 10 ina duplicate experiment.

FIG. 2C illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive 6 mg/kg of CFI-402257 daily for 21 days in a duplicateexperiment.

FIG. 2D illustrates change in CT26 tumor volume in Balb/cJ mice thatreceive the combination of 150 μg of rat IgG2a anti-PD-1 antibody onDays 0, 3, 6 and 10 and 6 mg/kg of CFI-402257 daily for 21 days in aduplicate experiment.

DETAILED DESCRIPTION

In one aspect, the present disclosure provides a method of treatingcancer in a subject, comprising the step of administering to the subjectan effective amount of the compound CFI-402257 that is represented bythe formula:

or a pharmaceutically acceptable salt thereof and an effective amount ofan immune checkpoint inhibitor.

It will be understood that unless otherwise indicated, theadministrations described herein include administering the describedcompound CFI-402257 or a pharmaceutically acceptable salt thereof priorto, concurrently with, or after administration of the immune checkpointinhibitor described herein. Thus, simultaneous administration is notnecessary for therapeutic purposes. In one embodiment, however, thecompound CFI-402257 or a pharmaceutically acceptable salt thereof isadministered concurrently with the immune checkpoint inhibitor.

The compound CFI-402257 described herein has basic amine groups andtherefore can form pharmaceutically acceptable salts withpharmaceutically acceptable acid(s). Accordingly, the term“pharmaceutically acceptable salt” as used herein refers to any suitablepharmaceutically acceptable acid addition salt of the compoundCFI-402257 described herein, which includes but is not limited to saltsof inorganic acids (e.g., hydrochloric acid, hydrobromic, phosphoric,nitric, and sulfuric acids) and of organic acids (such as, acetic acid,benzenesulfonic, benzoic, methanesulfonic, and p-toluenesulfonic acids).Other examples of such salts include hydrochlorides, hydrobromides,sulfates, methanesulfonates, nitrates, benzoates, fumarates, maleates,bisphosphate hemihydrates and salts with amino acids such as glutamicacid.

As used herein, an “immune check point inhibitor” or simply a“checkpoint inhibitor” refers to any compound that, either directly orindirectly, decreases the level of or inhibits the function of an immunecheckpoint receptor protein found on the surface of an immune cell(e.g., T-cells, B-cells, etc.). Alternatively, the immune checkpointinhibitor is a compound that, either directly or indirectly, decreasesthe level of or inhibits the function of a ligand on the surface of animmune cell-inhibitory cell (e.g., regulatory T-cells, tolerogenicantigen presenting cells (APC), myeloid-derived suppressor cells (MDSC),tumor-associated macrophages (TAM), cancer-assicatied fibroblasts (CAF),other cancer cells and APCs, etc.), or secreted by an immunecell-inhibitory cell. This ligand is typically capable of binding theimmune checkpoint receptor protein of the immune cell. A non-limitingexample of an immune checkpoint receptor protein-ligand pair isPD-1/PD-L1. PD-1 is an immune checkpoint receptor protein found onT-cells. PD-L1 that is often over-expressed by cancer cells binds toPD-1 and helps the cancer cells evade the host immune system attack.Accordingly, an immune checkpoint inhibitor prevents or reverses thisPD-1/PD-L1 binding, by either blocking the PD-1 on the T-cells (i.e., aPD-1 inhibitor) or the PD-L1 on the cancer cells (i.e., a PD-L1inhibitor), thereby maintaining or restoring anti-tumor T-cell activityor blocking T-cell-inhibitory cell activity. Additionally, an immunecheckpoint inhibitor refers to a compound as described in US PatentApplication Publication Nos. US 2017/0190675 and US 2016/0185870, andInternational Patent Application Publication Nos. WO 2015/112900, WO2010/027828 and WO 2010/036959.

An immune checkpoint inhibitor in accordance with the present inventionmay be a small-molecule organic compound or a large molecule such as apeptide or a nucleic acid. In at least one embodiment, an immunecheckpoint inhibitor is an antibody or an antigen binding fragmentthereof. In at least one embodiment, an immune checkpoint inhibitor is amonoclonal antibody or an antigen binding fragment thereof.

As used herein, the term “antibody” means an immunoglobulin moleculethat recognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. As usedherein, the term “antibody” encompasses intact polyclonal antibodies,intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)₂, and FV fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies, chimericantibodies, humanized antibodies, human antibodies, fusion proteinscomprising an antigen determination portion of an antibody, and anyother modified immunoglobulin molecule comprising an antigen recognitionsite so long as the antibodies exhibit the desired biological activity.An antibody can be of any of the five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and lgA2), based on the identity of theirheavy-chain constant domains referred to as alpha, delta, epsilon,gamma, and mu, respectively. The different classes of immunoglobulinshave different and well known subunit structures and three-dimensionalconfigurations. Antibodies can be naked or conjugated to other moleculessuch as toxins, radioisotopes, etc

In some embodiments, an antibody is a non-naturally occurring antibody.In some embodiments, an antibody is purified from natural components. Insome embodiments, an antibody is recombinantly produced. In someembodiments, an antibody is produced by a hybridoma.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and F_(v) fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. The term “antigen-binding fragment” of an antibody includesone or more fragments of an antibody that retain the ability tospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by certainfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of an antibodyinclude (without limitation): (i) an Fab fragment, a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and C_(H1) domains (e.g., anantibody digested by papain yields three fragments: two antigen-bindingFab fragments, and one Fc fragment that does not bind antigen); (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region (e.g., an antibodydigested by pepsin yields two fragments: a bivalent antigen-bindingF(ab′)₂ fragment, and a pFc′ fragment that does not bind antigen) andits related F(ab′) monovalent unit; (iii) a F_(d) fragment consisting ofthe V_(H) and C_(H1) domains (i.e., that portion of the heavy chainwhich is included in the Fab); (iv) a F_(v) fragment consisting of theV_(L) and V_(H) domains of a single arm of an antibody, and the relateddisulfide linked F_(v); (v) a dAb (domain antibody) or sdAb (singledomain antibody) fragment (Ward et al., Nature 341:544-546, 1989), whichconsists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR).

As used herein, a “monoclonal antibody” refers to a homogeneous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant, or epitope. This is in contrast topolyclonal antibodies that typically include different antibodiesdirected against different antigenic determinants. The term “monoclonalantibody” encompasses both intact and full-length monoclonal antibodiesas well as antibody fragments (such as Fab, Fab′, F(ab′)₂, F_(v)),single chain (scFv) mutants, fusion proteins comprising an antibodyportion, and any other modified immunoglobulin molecule comprising anantigen recognition site. Furthermore, “monoclonal antibody” refers tosuch antibodies made in any number of manners including but not limitedto by hybridoma, phage selection, recombinant expression, and transgenicanimals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., Nature 321:522-525,1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al.,Science 239:1534-1536, 1988).

A non-exhaustive list of examples of an immune checkpoint inhibitor isprovided as following: a CD40L inhibitor, a DR3 inhibitor, a TL1Ainhibitor, a GITR inhibitor, a GITRL inhibitor, a 4-1BB inhibitor, a4-1BBL inhibitor, an OX40 inhibitor, an OX40L inhibitor, a CD27inhibitor, a CD70 inhibitor, a TMIGD2 inhibitor, an HHLA2 inhibitor, anICOS inhibitor, an ICOSL inhibitor, a B7RP1 inhibitor, a CD28 inhibitor,a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4inhibitor, a CD80 inhibitor, a CD86 inhibitor, a KIR inhibitor, a TCRinhibitor, a LAG3 inhibitor, an MHCI inhibitor, an MHCII inhibitor, aCD80 inhibitor, a TIM-3 inhibitor, a GAL9 inhibitor, a BTLA inhibitor,an HVEM inhibitor, a CD160 inhibitor, a CD137 inhibitor, a CD137Linhibitor, a LIGHT inhibitor, a phosphatidylserine inhibitor, a VISTAinhibitor, a BTNL2 inhibitor, a B7-H3 inhibitor and a B7-H4 inhibitor.In certain embodiments, the immune checkpoint inhibitor applied in acancer treatment method of the invention is one or more selected fromthe aforementioned examples.

In one embodiment, the immune checkpoint inhibitor is one or moreselected from a PD-1 inhibitor, a PD-L1 inhibitor and a CTLA-4inhibitor.

In one embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor.In another embodiment, the immune checkpoint inhibitor is a PD-L1inhibitor.

In some embodiments, the immune checkpoint inhibitor is one or moreselected from pembrolizumab, ipilimumab, nivolumab, atezolizumab,avelumab and durvalumab.

In some embodiments, the immune checkpoint inhibitor is one or moreselected from JS001, SHR-1210, BGB-A317, 1B1-308, REGN2810, JS003,SHR-1316, KN035, BMS-936559, LAG525, BMS-986016, MBG453, MEDI-570,OREG-103/BY40 and lirilumab. In one embodiment, the immune checkpointinhibitor is one or more selected from CJS001, SHR-1210, BGB-A317,IBI-308 and REGN2810. In an alternative embodiment, the immunecheckpoint inhibitor is one or more selected from JS003, SHR-1316, KN035and BMS-936559.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, ameliorating, inhibiting or slowing theprogression of a cancer, reducing the likelihood of recurrence of acancer, or one or more symptoms thereof, as described herein. Exemplarytypes of cancer treated by the methods and compositions of the inventioninclude but are not limited to breast cancer (including metastaticbreast cancer); colon cancer; rectal cancer; colorectal cancer; lungcancer (including small-cell lung cancer (SCLC), non-small cell lungcancer (NSCLC), adenocarcinoma of the lung, and squamous carcinoma ofthe lung); cancer of the peritoneum; gastric or stomach cancer;gastrointestinal cancer; cervical cancer; liver cancer; bladder cancer;hepatoma; ovarian cancer; endometrial or uterine cancer; prostatecancer; testicular cancer; leukemias; lymphomas; hematologicalmalignancies; brain cancer (including glioma, glioblastoma multiforme,medulloblastoma, and neuroblastoma); head and neck cancer; pancreaticcancer; melanoma; hepatocellular cancer; kidney or renal cancer; vulvalcancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penilecarcinoma; Merkel cell cancer; mycoses fungoids; esophageal cancer;tumors of the biliary tract; salivary gland cancer; sarcomas;retinoblastoma; liposarcoma, synovial cell sarcoma; neuroendocrinetumors; gastrinoma; islet cell cancer; mesothelioma; schwannoma;acoustic neuroma; meningioma; adenocarcinoma; squamous cell cancer andepithelial squamous cell cancer. In another embodiment, the cancertreated by the methods and compositions of the invention is pancreaticcancer, lung cancer, breast cancer, colon cancer, brain cancer,neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme,ovarian cancer, lymphoma, leukemia, melanoma, sarcoma, paraneospasia,osteosarcoma, germinoma, glioma or mesothelioma. In yet anotherembodiment, the cancer is renal cell carcinoma, non-small cell lungcancer, urothelial cancer, head and neck cancer, ovarian cancer,lymphoma, melanoma, pancreatic cancer, myeloma, acute myeloid leukemia,bladder cancer and Hodgkin's lymphoma.

The term “an effective amount” means an amount when administered to asubject which results in beneficial or desired results, includingclinical results, i.e., reversing, alleviating, inhibiting or slowingthe progression of a cancer, reducing the likelihood of recurrence of acancer, or one or more symptoms thereof, e.g., as determined by clinicalsymptoms, the amount or volume or cancer cells or tumors in a subjectcompared to a control.

In an embodiment, an effective amount of the compound CFI-402257 or apharmaceutically acceptable salt thereof taught herein ranges from about0.1 to about 1000 mg/kg body weight, alternatively about 1 to about 500mg/kg body weight, and in another alternative, from about 1 to about 100mg/kg body weight, and in yet another alternative, from about 1 to about50 mg/kg. In an embodiment, an effective amount of an immune checkpointinhibitor taught herein ranges from about 0.01 to about 1000 μg persubject, alternatively from about 0.05 to about 500 μg per subject. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject suffering from cancer orreduce the likelihood of recurrence of a cancer. These factors include,but are not limited to, the classification and/or severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject and other diseases present.

In another aspect, pharmaceutical compositions comprising the compoundCFI-402257 or a pharmaceutically acceptable salt thereof and an immunecheckpoint inhibitor are also included in the present disclosure.

Also included are the use of the compound CFI-402257 or apharmaceutically acceptable salt thereof in the manufacture of amedicament to be used in combination with an immune checkpoint inhibitoras described herein for the treatment of one or more cancers describedherein. Also included herein are pharmaceutical compositions comprisingthe compound CFI-402257 or a pharmaceutically acceptable salt thereofand an immune checkpoint inhibitor as described herein optionallytogether with a pharmaceutically acceptable carrier, in the manufactureof a medicament for the treatment of one or more cancers describedherein. Also included is the compound CFI-402257 for use in combinationwith an immune checkpoint inhibitor as described herein for thetreatment of a subject with cancer. Further included are pharmaceuticalcompositions comprising the compound CFI-402257 or a pharmaceuticallyacceptable salt thereof and an immune checkpoint inhibitor as describedherein, optionally together with a pharmaceutically acceptable carrier,for use in the treatment of one or more cancers described herein.Further included are pharmaceutical compositions comprising the compoundCFI-402257 or a pharmaceutically acceptable salt thereof and an immunecheckpoint inhibitor as described herein optionally together with apharmaceutically acceptable carrier for use in the treatment of one ormore cancers described herein.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier, diluent, adjuvant, vehicle or excipient that does not adverselyaffect the pharmacological activity of the compound with which it isformulated, and which is also safe for human use. Pharmaceuticallyacceptable carriers that may be used in the compositions of thisdisclosure include, but are not limited to, ion exchangers, alumina,aluminum stearate, magnesium stearate, lecithin, serum proteins, such ashuman serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances (e.g.,microcrystalline cellulose, hydroxypropyl methylcellulose, lactosemonohydrate, sodium lauryl sulfate, and crosscarmellose sodium),polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycoland wool fat.

Other excipients, such as flavoring agents; sweeteners; andpreservatives, such as methyl, ethyl, propyl and butyl parabens, canalso be included. More complete listings of suitable excipients can befound in the Handbook of Pharmaceutical Excipients (5th Ed., aPharmaceutical Press (2005)). A person skilled in the art would know howto prepare formulations suitable for various types of administrationroutes. Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003, 20th edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999.

The compound CFI-402257 or a pharmaceutically acceptable salt thereofand the immune checkpoint inhibitor, or the compositions of the presentteachings may be administered, for example, by oral, parenteral,sublingual, topical, rectal, nasal, buccal, vaginal, transdermal, patch,pump administration or via an implanted reservoir, and thepharmaceutical compositions would be formulated accordingly. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationcan be by continuous infusion over a selected period of time.

Other forms of administration included in this disclosure are asdescribed in WO 2013/075083, WO 2013/075084, WO 2013/078320, WO2013/120104, WO 2014/124418, WO 2014/151142, and WO 2015/023915, thecontents of which are incorporated herein by reference.

EXEMPLIFICATION

While have described a number of embodiments of this, it is apparentthat our basic examples may be altered to provide other embodiments thatutilize the compounds and methods of this disclosure. Therefore, it willbe appreciated that the scope of this disclosure is to be defined by theappended claims rather than by the specific embodiments that have beenrepresented by way of example.

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

Example 1 Materials

Salt forms of the compound CFI-402257 were prepared using the one ormore of the procedures described in U.S. Pat. No. 9,657,025,International Publication Application Nos. WO 2014/075168 and WO2015/070349, and Liu et al. (2016) ACS Med Chem Lett 7(7):671-675. Thehydrochloride or bisphosphate hemihydrate salt forms of the compoundCFI-402257 were used in all of the studies described herein.

Rat IgG2a anti-PD-1 antibody (clone RMP1-14; cat. no. BE0146) and ratIgG2a isotype control (clone 2A3; cat. no. BE0089) were obtained fromBio X Cell.

BALB/cJ mice were obtained from The Jackson Laboratory. Six- toeight-week-old female animals were used for all of the studies describedherein and were allowed unrestricted access to food and water. Allanimal procedures were approved by the institutional animal care and usecommittee of the University Health Network (Toronto).

The CT26 mouse colon carcinoma cell line was obtained from American TypeCulture Collection (ATCC) and maintained according to the supplier'sinstructions. Short tandem repeat (STR) profiling was used to verifyauthenticity of the cell lines. Sixteen STR loci were simultaneouslyamplified in multiplex PCR at The Centre for Applied Genomics (Toronto),and the ATCC database was used for comparison when possible Cell lineswere routinely tested for mycoplasma and used at low passage numbers(<15).

Example 2 Methods

BALB/cJ mice were inoculated subcutaneously with 1×10⁶ CT26 cells. Themice were then randomized. Animal weights were monitored daily, andtumor volume was measured three times per week.

Tumor volume (in cubic millimeters or mm³) was defined as100×└1−TV_(f, treated)−TV_(i, treated))/(TV_(f, control)−TV_(i, control))],where TV_(f) is the average tumor volume at the end of study and TV, isthe average tumor volume at the end initiation of treatment. In cases inwhich tumor regression occurred, percentage of tumor regression wasdefined as 100×[1−(TV_(f, treated)/TV_(i, treated))]. At the completionof each study, the mice were killed by an anesthetic overdose, and tumortissue was removed for further analysis.

Treatments were initiated when tumor volumes reached an average size of˜60 mm³. To treat an established tumor, the animals were first assignedinto groups, i.e., the control group receiving rat IgG2a PD-1 antibody,the group receiving the compound CFI-402257 monotherapy, the groupreceiving the rat IgG2a anti-PD-1 antibody monotherapy and the groupreceiving the combination therapy. The compound CFI-402257 and thevehicle [10% (v/v) NMP/40% (v/v) PEG-300/50% (v/v) water for thehydrochloride form; or 80-90% (v/v) PEG-300 or PEG-400 20-10% (v/v)water for the bisphosphate hemihydrate form] were administered by oralgavage (PO), at 6 mg/kg daily (QD) for 21 days. The anti-PD-1 antibodyor the isotype control were administered by intraperitoneal (IP)injection. 150 μg anti-PD-1 antibody was administered in four doses,i.e., on Days 0, 3, 6 and 10. These treatment studies were carried outin duplicates.

Example 3

Complete Regression in Combination CFI-402257 andAnti-PD-1-Antibody-Treated Tumors

The size of each individual tumor within each treatment arm is plotted(See FIGS. 1A-1D and 2A-2D). As can be seen in FIGS. 1A and 2A, tumorsin the vehicle-treated control arm grew rapidly, and the average tumorwas >1500 mm³ by Day 11 of treatment.

As shown in FIGS. 1B and 2B, there was tumor growth delay in theanti-PD-1-antibody-treated single agent arms. Similarly, theCFI-402257—treated single agent arm in FIGS. 1C and 2C indicate tumorgrowth delay and that the administered dosage was well-tolerated.Although there was tumor growth inhibition in both monotherapeutic arms,there were no instances in which complete regression was observed.

Surprisingly and notably, in the combination anti-PD-1 antibody andCFI-402257-treated arm of FIG. 1D, two of the eight tumors completelyregressed. Very similar results were achieved in the duplicateexperiment of FIG. 2D, where complete regression was again observed intwo of eight tumors.

Example 4 CFI-402257 and anti-PD-1-antibody generates tumor immunity

Furthermore, in the first experiment, the two animals in which completeregression had occurred were re-challenged by inoculation with CT26cells on Day 31. Tumors did not grow in either mouse, indicating thatimmunity to the CT26 cells had been generated.

Example 5 Animal Body Weight Monitoring

In the first experiment, none of the animals receiving the CFI-402257 oranti-PD-1-antibody single agent treatment or the combination treatmentrecorded any significant body weight loss. Limited body weight loss(7.5%) was observed only in animals treated with only CFI-402257 in thesecond experiment.

While the applicants have described a number of embodiments of thisinvention, it is apparent that these basic examples may be altered toprovide other embodiments that utilize the compounds and methods of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments that have been represented by way of example.

1. A method for treating colon cancer, comprising: administering to asubject an effective amount of a compound represented by the formula:

or a pharmaceutically acceptable salt thereof and an effective amount ofa PD-L1 inhibitor.
 2. The method according to claim 1, wherein the PD-L1inhibitor is an antibody or an antigen binding fragment thereof.
 3. Themethod according to claim 1, wherein the PD-L1 inhibitor is a monoclonalantibody or an antigen binding fragment thereof. 4-7. (canceled)
 8. Themethod according to claim 1, wherein the PD-L1 inhibitor restoresanti-tumor T-cell activity.
 9. The method according to claim 1, whereinthe PD-L1 inhibitor blocks T-cell-inhibitory cell activity.
 10. Themethod according to claim 1, wherein the PD-L1 inhibitor is one selectedfrom atezolizumab, avelumab and durvalumab. 11-12. (canceled)
 13. Themethod according to claim 1, wherein the PD-L1 inhibitor is one selectedfrom JS003, SHR-1316, KN035 and BMS-936559. 14-16. (canceled)
 17. Apharmaceutical composition comprising a compound represented by theformula:

or a pharmaceutically acceptable salt thereof and a PD-L1 inhibitor. 18.The pharmaceutical composition according to claim 17, wherein the PD-L1inhibitor is an antibody or an antigen binding fragment thereof.
 19. Thepharmaceutical composition according to claim 17, wherein the PD-L1inhibitor is a monoclonal antibody or an antigen binding fragmentthereof. 20-23. (canceled)
 24. The pharmaceutical composition accordingto claim 17, wherein the PD-L1 inhibitor restores anti-tumor T cellactivity.
 25. The pharmaceutical composition according to claim 17,wherein the PD-L1 inhibitor blocks T-cell-inhibitory cell activity. 26.The pharmaceutical composition according to claim 17, wherein the PD-L1inhibitor is one selected from atezolizumab, avelumab and durvalumab.27-28. (canceled)
 29. The pharmaceutical composition according to claim17, wherein the L1 inhibitor is one selected from JS003, SHR-1316, KN035and BMS-936559.